Thermostable DNA polymerases have been disclosed previously as set forth in U.S. Pat. No. 5,192,674 to Oshima et al., U.S. Pat. Nos. 5,322,785 and 5,352,778 to Comb et al., U.S. Pat. No. 5,545,552 to Mathur, and others. All of the noted references recite the use of polymerases as important catalytic tools in the practice of molecular cloning techniques such as polymerase chain reaction (PCR). Each of the references states that a drawback of the extant polymerases are their limited thermostability, and consequent useful life in the participation in PCR. Such limitations also manifest themselves in the inability to obtain extended lengths of nucleotides, and in the instance of Taq polymerase, the lack of 3′ to 5′ exonuclease activity, and the drawback of the inability to excise misinserted nucleotides (Perrino, 1990).
More generally, such polymerases, including those disclosed in the referenced patents, are of the Polymerase I variety as they are often 90-95 kDa in size and may have 5′ to 3′ exonuclease activity. They define a single subunit with concomitant limits on their ability to hasten the amplification process and to promote the rapid preparation of longer strands of DNA.
Chromosomal replicases are composed of several subunits in all organisms (Kornberg and Baker, 1992). In keeping with the need to replicate long chromosomes, replicases are rapid and highly processive multiprotein machines. Cellular replicases are classically comprised of three components: a clamp, a clamp loader, and the DNA polymerase (reviewed in Kelman and O'Donnell, 1995; McHenry, 1991). For purposes of the present invention, the foregoing components also serve as a broad definition of a “Pol III-type enzyme”.
DNA polymerase III holoenzyme (Pol III holoenzyme) is the multi-subunit replicase of the E. coli chromosome. Pol III holoenzyme is distinguished from Pol I type DNA polymerases by its high processivity (>50 kbp) and rapid rate of synthesis (750 nts/s) (reviewed in Kornberg and Baker, 1992; Kelman and O'Donnell, 1995). The high processivity and speed is rooted in a ring shaped subunit, called β, that encircles DNA and slides along it while tethering the Pol III holoenzyme to the template (Stukenberg et al., 1991; Kong et al., 1992). The ring shaped β clamp is assembled around DNA by the multisubunit clamp loader, called γ complex. The γ complex couples the energy of ATP hydrolysis to the assembly of the β clamp onto DNA. This γ complex, which functions as a clamp loader, is an integral component of the Pol III holoenzyme particle. A brief overview of the organization of subunits within the holoenzyme and their function follows.
Pol III holoenzyme consists of 10 different subunits, some of which are present in multiple copies for a total of 18 polypeptide chains (Onrust et al., 1995). The organization of these subunits in the holoenzyme particle is illustrated in FIG. 1. As depicted in the diagram, the subunits of the holoenzyme can be grouped functionally into three components: 1) the DNA polymerase III core is the catalytic unit and consists of the α (DNA polymerase), ε (3′-5′ exonuclease), and θ subunits (McHenry and Crow, 1979), 2) the β “sliding clamp” is the ring shaped protein that secures the core polymerase to DNA for processivity (Kong et al., 1992), and 3) the 5 protein γ complex (γδδ′χψ) is the “clamp loader” that couples ATP hydrolysis to assembly of β clamps around DNA (O'Donnell, 1987; Maki et al., 1988). A dimer of the τ subunit acts as a “macromolecular organizer” holding together two molecules of core (Studwell-Vaughan and O'Donnell, 1991; Low et al., 1976) and one molecule of γ complex forming the Pol III* subassembly (Onrust et al., 1995). This organizing role of τ to form Pol III* is indicated in the center of FIG. 1. Two β dimers associate with the two cores within Pol III* to form the holoenzyme, which is capable of replicating both strands of duplex DNA simultaneously (Maki et al., 1988).
The DNA polymerase III holoenzyme assembles onto a primed template in two distinct steps. In the first step, the γ complex assembles the β clamp onto the DNA. The γ complex and the core polymerase utilize the same surface of the β ring and they cannot both utilize it at the same time (Naktinis et al., 1996). Hence, in the second step the γ complex moves away from β thus allowing access of the core polymerase to the β clamp for processive DNA synthesis. The γ complex and core remain attached to each other during this switching process by the τ subunit organizer.
The γ complex consists of 5 different subunits (γ2-4δ1δ′1χ1ψ1). An overview of the mechanism of the clamp loading process follows. The δ subunit is the major touch point to the β clamp and leads to ring opening, but δ is buried within γ complex such that contact with β is prevented (Naktinis et al., 1995). The γ subunit is the ATP interactive protein but is not an ATPase by itself (Tsuchihashi and Kornberg, 1989). The δ′ subunit bridges the δ and γ subunits resulting in a γδδ′ complex that exhibits DNA dependent ATPase activity and is competent to assemble clamps on DNA (Onrust et al., 1991). Upon binding of ATP to γ, a change in the conformation of the complex exposes δ for interaction with β (Naktinis et al., 1995). The function of the smaller subunits, χ and ψ, is to contact SSB (through χ) thus promoting clamp assembly and high processivity during replication (Kelman and O'Donnell, 1995).
The three component Pol III-type enzyme in eukaryotes contains a clamp that has the same shape as E. coli β, but instead of a homodimer it is a heterotrimer. This heterotrimeric ring, called PCNA (proliferating cell nuclear antigen), has 6 domains like β, but instead of each PCNA monomer being composed of 3 domains and dimerizing to form a 6 domain ring (e.g., like β), the PCNA monomer has 2 domains and it trimerizes to form a 6 domain ring (Krishna et al., 1994; Kuriyan and O'Donnell, 1993). The chain fold of the domains are the same in prokaryotes (β) and eukaryotes (PCNA); thus, the rings have the same overall 6-domain ring shape. The clamp loader of the eukaryotic Pol III-type replicase is called RFC (Replication factor C) and it consists of subunits having homology to the γ and δ′ subunits of the E. coli γ complex (Cullmann et al., 1995). The eukaryotic DNA polymerase III-type enzyme contains either of two DNA polymerases, DNA polymerase δ and DNA polymerase ε (Bambara and Jessee, 1991; Linn, 1991; Sugino, 1995). It is entirely conceivable that yet other types of DNA polymerases can function with either a PCNA or β clamp to form a Pol III-type enzyme (for example, DNA polymerase II of E. coli functions with the β subunit placed onto DNA by the γ complex clamp loader) (Hughes et al., 1991; Bonner et al., 1992). The bacteriophage T4 also utilizes a Pol III-type 3-component replicase. The clamp is a homotrimer like PCNA, called gene 45 protein (Young et al., 1992). The gene 45 protein forms the same 6-domain ring structure as β and PCNA (Moarefi et al., 2000). The clamp loader is a complex of two subunits called the gene 44/62 protein complex. The DNA polymerase is the gene 43 protein and it is stimulated by the gene 45 sliding clamp when it is assembled onto DNA by the 44/62 protein clamp loader. The Pol III-type enzyme may be either bound together into one particle (e.g., E. coli Pol III holoenzyme), or its three components may function separately (like the eukaryotic Pol III-type replicases).
There is an early report on separation of three DNA polymerases from T.th. cells, however each polymerase form was reminiscent of the preexisting types of DNA polymerase isolated from thermophiles in that each polymerase was in the 110,000-120,000 range and lacked 3′-5′ exonuclease activity (Ruttimann et al., 1985). These are well below the molecular weight of Pol III-type complexes that contain in addition to the DNA polymerase subunit, other subunits such as γ and τ. Although the three polymerases displayed some differences in activity (column elution behavior, and optimum divalent cation, template, and temperatures) it seems likely that these three forms were either different repair type polymerases or derivatives of one repair enzyme (e.g., Pol I) that was modified by post translational modification(s) that altered their properties (e.g. phosphorylation, methylation, proteolytic clipping of residues that alter activity, or association with different ligands such as a small protein or contaminating DNA). Despite this previous work, it remained to be demonstrated that thermophiles harbor a Pol III-type enzyme that contain multiple subunits such as γand/or τ, functioned with a sliding clamp accessory protein, or could extend a primer rapidly and processively over a long stretch (>5 kb) of ssDNA (Ruttimann et al., 1985).
Previously, it was not known what polymerase thermophilic bacteria used to replicate their chromosome since only Pol I type enzymes have been reported from thermophiles. By distinction, chromosomal replicases, such as Polymerase III, identified in E. coli, if available in a thermostable bacterium, with all its accessory subunits, could provide a great improvement over the Polymerase I type enzymes, in that they are generally much more efficient—about 5 times faster—and much more highly processive. Hence, one may expect faster and longer chain production in PCR, and higher quality of DNA sequencing ladders. Clearly, the ability to practice such synthetic techniques as PCR would be enhanced by these methods disclosed for how to obtain genes and subunits of DNA polymerase III holoenzyme from thermophilic sources.
The present invention is directed to achieving these objectives and overcoming the various deficiencies in the art.